This invention relates to a method for maturing human dendritic cells, to a method for enhancing production of interleukin 12 (IL 12) from mature dendritic cells, and to a vaccine containing matured dendritic cells producing enhanced levels of IL 12.
It has, been established that cytotoxic CD8+ T-cell lymphocytes (CTL) can recognize and kill tumour cells which present tumour antigens on the cell surface in conjunction with MHC (major histocompatability complex) class I molecules(1). However, in the majority of patients who are diagnosed with cancer, the patient's cellular immune response is not sufficiently activated in response to tumour antigens, and therefore the patient's body is unable to adequately kill the tumour cells and so defend itself from the further spread of the cancer. Patients may therefore be treated with chemotherapy or radiation therapy, both of which may indiscriminately kill the normal cells and may cause significant toxic side effects to the patient. If the cellular immune response could be sufficiently activated by tumour antigens, then it is possible that the patient's body would be able to eradicate the tumour cells itself, without the undesirable side effects associated with conventional types of cancer treatment.
Therefore, a need exists to therapeutically activate a cancer patient's cellular immune system so that it responds to tumour-associated antigens.
Dendritic cells are among the most powerful antigen-presenting cells for priming both CD8+ cytotoxic T-cells (CTL) and CD4+ T-helper (Th1) responses(2). They are capable of capturing and processing antigens and migrating to the regional lymph nodes to induce CD8+ T-cell responses(2). They have the capacity to cross-present exogenous antigens in the context of MHC class I molecules present on the cell surface(3). These features taken together enable the dendritic cells to present antigen in a manner which is capable of priming both CD8+ and CD4+ T-ell responses, providing a rationale for the use of dendritic cells as a cellular vaccine. However, for this it is necessary to have dendritic cells available in sufficient numbers and in a functionally optimum antigen loaded state.
Murine studies have supported the immunizing capacities of bone marrow-derived dendritic cells propagated in vitro with GM-CSF and interleukin 4 (IL 4), and pulsed with the relevant CTL defined tumour associated epitopes.(4,5) The studies have demonstrated that dendritic cells primed with defined tumour associated antigen peptides are capable of eradicating established tumours expressing the appropriate tumour antigens. These dendritic cell-mediated anti-tumour responses in animal models have been shown to be dependent on CD4+ T-helper (MHC class II) as well as CD8+ (MHC class I) responses and also on the production of Th1 lymphokines(5).
These animal studies have led to a number of phase 1 human clinical trials using mature and immature autologous dendritic cells loaded with tumour antigens. For example, Nestle et al(6) have treated 16 patients with metastatic melanoma with immature GM-CSF/IL 4 monocyte-derived dendritic cells grown in fetal calf serum. Clinical response was seen in 5 out of 16 patients usually durable (2 complete responses and 3 partial responses) with skin, soft tissue, lung and pancreatic metastases. Monocyte-derived dendritic cells pulsed with MAGE-3 tumour specific peptides and matured with TNF-α similarly induced responses in 6 out of 11 patients with skin, lymph node, lung and liver metastases(7). A significant expansion of MAGE-3 HLA A1-specific CD8+ T-cells was observed in 6 out of 11 patients and response of skin metastases was associated with a CD8+ T-cell infiltrate.
Evidence supporting the efficacy of dendritic cells as immunotherapeutic agents has also been gathered from clinical trials involving patients with metastatic cancers from other types of primary tumours. Immunisation with dendritic cells prepared from the fusion of allogeneic monocytes and autologous tumour cells, and matured with TNF-α, were successful in inducing cellular immune responses in 7 out of 11 patients with metastatic renal cell carcinoma, including 4 complete remissions(8). GM-CSF/IL 4 immature dendritic cells pulsed with prostate membrane antigen P1 and P2 have been employed in 37 patients with advanced prostate cancer. One complete response and 10 partial responses (>50% reduction in PSA levels or significant resolution on a bone scan) were observed(9). In a series of 9 patients(10) with advanced cervical cancer who were treated with immature GM-CSF/IL 4 dendritic cells pulsed with allogeneic HPV 16+ve tumour lysate specific HPV specific CTL, response was demonstrated in peripheral blood in 2 out of 2 evaluable (HPV16+HLA 002*) patients after vaccination. In one patient the frequency of HPV16E7 (11-20) rose to 2.2% as detected by class1 tetramers and in the other patient the IFN-γ ELISPOT assay revealed a specific response to 4 HPV 16 E6 and 7 derived CTL epitopes, 1 week and 2 months, respectively, after vaccination. In 1 out of 4 evaluable HPV 16+ patients a specific T-helper response was also observed. T cell immunity as detected by ELISPOT correlated with the DTH response to tumour lysate and these patients followed a favourable clinical outcome (NED of disease 18 months or more after resection of lung metastasis, stable disease for 3 months or more after progression).
Therefore it is feasible to induce clinically relevant specific class I and T-helper responses in patients with metastases from a variety of cancer types using monocyte-derived dendritic cells pulsed with a variety of tumour associated antigens. However, currently no consensus exists with respect to the definition of the immunologically active phenotype, dose, route and loading method for optimum cancer immunization with dendritic cells(11).
It seems mature dendritic cells are likely to be more effective at presenting antigens and triggering CTL and T-helper response than immature dendritic cells(2). Whilst clinical anti-cancer responses have been observed following immunization with immature dendritic cells it is likely that in these patients dendritic cells may have been at some point matured in vivo by an as yet undefined stimulus. Dendritic cells normally acquire antigens from peripheral tissues in their immature state. Maturation Is characterized by downregulation of their antigen-acquisition capacity, increased expression of MHC and co-stimulatory molecules on their surface, raised level of IL 12 production by them, and altered expression of chemokine receptors(12).
Thus a means for deliberate maturation of dendritic cells in vitro prior to their use for vaccination may offer the advantage of a phenotype with an optimum migratory capacity to lymph nodes to prime T-cells in lymph nodes, an optimum Th1 lymphokine(13) production capacity as well as a stable functional state which is least susceptible to the cancer associated tolerogenic influences such as Interleukin 10(14).
Critical to whether the T-cells are activated or energised by interaction with dendritic cells, appears to be the nature of the “activation” or “danger signal”, which may be pathogen-induced or triggered by factors released by stressed, damaged or necrotic cells as originally proposed by Matzinger(15). However the nature of the optimum “activation” or “danger signal” still remains to be defined, though in vitro data appear to suggest that whatever its ultimate nature it requires to be able to induce maturation of dendritic cells and IL 12 production by the dendritic cells, two properties which are important for optimum CD8+ T-cell response.
Bacterial DNA, CD40 ligand, pro-inflammatory agents such as LPS, viral infections, CpG-oligodeoxynucleotides and heatshock proteins can all initiate maturation of dendritic cells(16-19). Lymphokines such as TNF-α and type 1 interferons are also known to induce reversible maturation of the dendritic cells(20-21). In contrast, the supernatant of activated monocytes (monocyte-derived medium) appears to be an agent capable of inducing a stable maturation state, but it is difficult to standardize its quality for clinical use(22). For clinical immunotherapeutic application of dendritic cells, a stable dendritic cell phenotype which produces high levels of biologically active IL 12, appears to be ideal
Poly [I]: poly [C] (polyriboinosinlc:polyribocytidylic acid), a synthetic dsRNA (double stranded RNA), has been found to induce a stable mature phenotype with high expression levels of CD86 and the maturation marker CD83. The mature phenotype is retained for 48 hours after cytokine withdrawal and these mature dendritic cells produce high levels of IL 12 and low levels of IL 10(23). Activation of dendritic cells with a microbial stimulus (e.g. CpG oligonucleotides) and a range of bacterial stimuli in the absence of a T-cell derived signal appears to be sufficient to release significant levels of IL 12(24). Under these conditions dendritic cells upregulate CD40 expression and subsequent cross-linking of CD40 can result in further enhanced IL 12 production(24). The notion that optimal production of IL 12 p70 by dendritic cells involves synergy between CD40 cross-linking and microbial stimulation is compatible with human in vitro studies that demonstrate that the interaction between T-cells and antigen-presenting cells is not sufficient to induce high levels of IL 12 production unless microbial stimuli and/or cytokines are used as the adjuvants, or alternatively IL 12 production is induced by the interaction of the dendritic cells with the T-cells(25-26). These data emphasize the importance of bacterial stimuli in the production of high levels of IL 12 by dendritic cells. In addition these data suggest that the potency of CD40 mABs(27-29) might be augmented by co-administration of an appropriate bacterial adjuvant in immunotherapy.
For clinical immunotherapeutic application there is a need for a nontoxic and clinical grade stimulus capable of inducing dendritic cells to produce maximum levels of IL 12 when dendritic cells are used as a cellular vaccine. Alternatively, the stimulus should be capable of being applied as a potential systemic adjuvant to a vaccine, which requires the promotion of a Th1 effect. Poly [I]: poly [C] in doses of up to 75 mg/m2 intravenously as well as its various stable derivatives (principally dsRNA complexes with polylysine or cellulose) were tested in the 1970s and 1980s in a number of phase 1 and 2 anti-cancer trials. However, these trials had to be abandoned because of the toxic effects of poly[I]: poly[C], which included shock, renal failure and coagulopathies and hypersensitivity reactions(30-32).
Modifications in the structural characteristics of poly[I]:poly [C] by the introduction of unpaired bases (uracil and guanine) has resulted in unique dsRNAs, termed “specifically configured dsRNAs” or “mismatched dsRNAs” (33). These regions appear to accelerate dsRNA hydrolysis and reduce toxicity in humans (34) whilst retaining ability to promote interferon synthesis. AMPLIGEN® (poly[I]:poly[C12U]) is one such synthetic dsRNA containing regularly occurring regions of mismatching (non-hydrogen bonding) along the helical dsRNA chain. AMPLIGEN ® (poly[I]:poly[C12U]) exerts immunoregulatory activity, antiviral activity against RNA and DNA viruses and tumour cell antiproliferative activity in vitro and in vivo(33).
Clinical experience with AMPLIGEN® (poly[I]:poly[C12U]) currently totals more than 300 patients. No evidence of dose-limiting organ toxicity, including hematological, liver or renal toxicity, has been observed and AMPLIGEN® (poly [I]:poly [C12U]) is prepared under GMP conditions for clinical use(34).
The term “specifically configured” as used herein is intended to refer to a double stranded RNA which contains regularly occurring regions of mismatched bases. As there are no hydrogen bonds between the mismatched bases, the double helix is weakened. The half-life of the dsRNA is therefore reduced because it is more easily and quickly degraded, making the dsRNA less toxic to humans and animals.